JP2022544698A - Composition for prevention or treatment of non-alcoholic steatohepatitis containing exosomes derived from induced pluripotent stem cell-derived mesenchymal stem cell progenitor cells - Google Patents

Abstract

The present invention provides a preventive or therapeutic agent for non-alcoholic steatohepatitis, which contains exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells pretreated or not pretreated with a pretreatment substance as an active ingredient. relating to scientific compositions. The exosomes of the present invention exhibit improved preventive or therapeutic effects on non-alcoholic steatohepatitis compared to exosomes isolated from conventional mesenchymal stem cells, and are useful for related research and development and commercialization. Available. [Selection drawing] Fig. 17A

Description

This patent application is based on Korean Patent Application No. 10-2019-0103186 filed with the Korean Patent Office on August 22, 2019, and Korean Patent Application No. 10-2020- filed with the Korean Intellectual Property Office on August 4, 2020. 0097398, the disclosures of which are incorporated herein by reference.

The present invention provides a nonalcoholic fatty exosome containing, as an active ingredient, exosomes derived from mesenchymal stem cells differentiated from induced pluripotent stem cell-derived mesenchymal stem progenitor cells pretreated or not pretreated with a pretreatment substance. It relates to a composition for prevention or treatment of hepatitis.

Mesenchymal stem cells are pluripotent stromal cells and refer to cells capable of differentiating into various cells including osteoblasts, chondrocytes, muscle cells, adipocytes and the like. Mesenchymal stem cells can differentiate into various connective tissues such as cartilage, bone tissue, ligaments, and bone marrow stromal cells. It is being studied as a therapeutic use for

Recently, he has been actively researching therapeutic effects for various diseases using exosomes secreted by mesenchymal stem cells, without using mesenchymal stem cells themselves. For commercial use of such exosomes, large quantities and high quality of exosomes are required. However, the amount of exosomes obtained from mesenchymal stem cells is very small, and the function and proliferative ability of mesenchymal stem cells also decrease with repeated passages. There is an emerging need for the development of a technique for establishing cells that are highly proliferative while possessing the ability to grow.

Non-alcoholic steatosis, on the other hand, is characterized by accumulation of triglycerides in hepatocytes despite the absence of excessive alcohol intake. Non-alcoholic steatosis continues to increase due to excess nutrition associated with high fat and high carbohydrate intake in modern humans. Non-alcoholic steatosis is commonly observed in obesity and diabetes, and various factors are known to be associated with non-alcoholic steatosis. It has been reported that 80% of adults with non-alcoholic steatosis develop metabolic diseases such as insulin-resistant diabetes and heart disease.

Non-alcoholic steatosis is classified into non-alcoholic simple steatosis and non-alcoholic steatohepatitis (NASH) with inflammation, but long-term Left untreated, it can lead to serious liver disease, including hepatitis, liver fibrosis, and cirrhosis. Nonalcoholic steatosis is characterized by the accumulation of fat (fatty infiltration) in the hepatocytes.
Non-alcoholic simple fatty liver may progress to non-alcoholic steatohepatitis. In nonalcoholic steatohepatitis, fat accumulation is known to be associated with varying degrees of liver inflammation and hepatic scarring, often associated with insulin resistance, dyslipidemia, and hypertension. there is Non-alcoholic steatohepatitis often develops from people with excess weight, excess blood cholesterol and triglyceride levels, and/or insulin resistance.

In recent years, as the number of patients with non-alcoholic steatohepatitis has increased along with the increase in the obese population, the therapeutic agent market for non-alcoholic steatohepatitis has developed on a large scale. Elucidation of the cause and mechanism of non-alcoholic steatohepatitis has attracted strong interest in the development of therapeutic agents for non-alcoholic steatohepatitis. However, the development of safe, long-term therapeutic agents for non-alcoholic steatohepatitis is still very limited.
For the treatment of non-alcoholic steatohepatitis, anti-obesity drugs, insulin resistance drugs, hyperlipidemia drugs, hepatoprotective agents, antioxidants and the like are commonly used. However, these drugs are not essential therapeutic agents for non-alcoholic steatohepatitis, but are used as symptom improving agents and have side effects when taken for a long period of time.
Therefore, there is an increasing demand for the development of a new therapeutic composition that is safer, can be administered over a long period of time, and is suitable for the treatment of non-alcoholic steatohepatitis, a chronic disease.

As a result of intensive and thorough research on the development of therapeutic agents for non-alcoholic steatohepatitis using mesenchymal stem cell exosomes, the present inventors found that induced pluripotent stem cells differentiated from progenitor cells. cell; iPSC)-derived mesenchymal stem cells are established, and induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells differentiated from the progenitor cells pretreated with a pretreatment substance or not pretreated. Exosomes have been found to exhibit excellent preventive and therapeutic effects on alcoholic steatohepatitis.

Accordingly, an object of the present invention is to provide non-alcoholic steatohepatitis containing exosomes isolated from induced pluripotent stem cells (iPSCs)-derived mesenchymal stem cells (MSCs) as active ingredients. (Non-alcoholic steatohepatitis; NASH).
Another object of the present invention is to provide exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells.
Still another object of the present invention is to provide a non-alcoholic fatty exosome containing as an active ingredient exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells (MSCs) pretreated with a pretreatment substance. An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis.
Yet another object of the present invention is to provide exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells pretreated with a pretreatment substance.

The present inventors conducted intensive and thorough research on the development of therapeutic agents for non-alcoholic steatohepatitis using mesenchymal stem cell exosomes. As a result, mesenchymal stem cells derived from induced pluripotent stem cells (iPSCs) were established by differentiation of their progenitor cells, and the induced pluripotent stem cells were pretreated with or without a pretreatment substance. Exosomes derived from mesenchymal stem cells derived from potent stem cells were found to exhibit excellent preventive or therapeutic effects on non-alcoholic steatohepatitis.
The present invention provides exosomes isolated from mesenchymal stem cells differentiated from progenitor cells of induced pluripotent stem cell-derived mesenchymal stem cells, and pharmaceuticals for the prevention or treatment of nonalcoholic steatohepatitis containing the exosomes as active ingredients. and exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells pretreated with a pretreatment substance, and a pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis containing the same as an active ingredient Regarding the composition.

The present invention will now be described in more detail.
One aspect of the present invention includes exosomes isolated from induced pluripotent stem cells (iPSCs)-derived mesenchymal stem cells (MSCs) as active ingredients for non-alcoholic steatohepatitis ( The present invention relates to a pharmaceutical composition for prevention or treatment of Non-alcoholic steatohpatitis.
Another aspect of the present invention is a pharmaceutical composition for preventing, alleviating, improving, or treating non-alcoholic steatohepatitis, comprising exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells as an active ingredient. Regarding.

The term “nonalcoholic steatohepatitis” as used herein is one of nonalcoholic fatty liver disease (NAFLD), a progressive disease characterized by fatty liver accompanied by inflammation or fibrosis. It is a liver disease and means a precursor disease that induces cirrhosis and liver cancer.

As used herein, the term "stem cell" refers to an undifferentiated cell that has the capacity to self-renew and differentiate into two or more different cell types. Stem cells of the invention may be autologous stem cells or homologous stem cells.

As used herein, the term "induced pluripotent stem cells" refers to cells that have been reprogrammed to an undifferentiated pluripotency state by inducing dedifferentiation into already differentiated cells such as somatic cells. means that
The dedifferentiation is performed by introducing and expressing a specific gene (e.g., Sox2, c-Myc, Klf4, Oct-4, etc.), or a dedifferentiation-inducing protein expressed in cells into which the specific gene has been introduced. can be injected and induced.
The pluripotency means the ability to differentiate into a tissue or organ of any origin of the three germ layers, endoderm, mesoderm, and ectoderm. do.

As used herein, the term "mesenchymal stem cells" refers to pluripotent cells capable of differentiating into various cells including osteocytes, chondrocytes, muscle cells, adipocytes, and the like. As for the mesenchymal stem cells, bone marrow-derived mesenchymal stem cells are most frequently used, but in addition to bone marrow, umbilical cord or cord blood, adipose tissue, amniotic fluid, and tooth buds of molars are also used. good too. Mesenchymal stem cells are also called stromal cells.
The mesenchymal stem cell progenitor cells are not general mesenchymal stem cell progenitor cells, but mesenchymal cells derived from induced pluripotent stem cells (iPSCs) (developed by the present inventors). They are progenitor cells of stem cells.

In the present invention, progenitor cells of induced pluripotent stem cell-derived mesenchymal stem cells may not express SSEA-4 (stage-specific embryonic antigen 4) protein.
In the present invention, induced pluripotent stem cell-derived mesenchymal stem cells are differentiated from progenitor cells of induced pluripotent stem cell-derived mesenchymal stem cells that do not express SSEA-4 (stage-specific embryonic antigen 4) protein. you can

The "induced pluripotent stem cells" refer to cells that have been induced to have pluripotent differentiation potential by an artificial reprogramming process from differentiated cells, and are also referred to as reprogrammed stem cells.
Artificial reprogramming processes are by introduction of viral-mediated reprogramming factors using retroviruses, lentiviruses, and Sendai viruses, or non-viral-mediated reprogramming factors using non-viral vectors, proteins, cell extracts, and the like. done. Reprogramming processes also include reprogramming with stem cell extracts, compounds, and the like.
Induced pluripotent stem cells have almost the same characteristics as embryonic stem cells, in particular exhibit similar cell morphology, similar gene and protein expression patterns, and have pluripotency in vitro and in vivo, Forming a teratoma and inserting it into a mouse blastocyst forms a chimera mouse, allowing germline transmission of genes.
The induced pluripotent stem cells of the present invention include all mammal-derived induced pluripotent stem cells such as humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice, and rabbits, but preferably human-derived induced pluripotent stem cells. It is an induced pluripotent stem cell.
In addition, the somatic cells before reprogramming by the induced pluripotent stem cells of the present invention are somatic cells derived from umbilical cord, cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, amniotic fluid, placenta, or the like. but not limited to this. Specifically, the somatic cells include fibroblasts, hepatocytes, adipose cells, epithelial cells, epidermal cells, chondrocytes, muscle cells, cardiac muscle cells, melanocytes, neural cells, glial cells, astroglial cells, monocytes, macrophages (macrophage) and the like, but are not limited thereto.

In one embodiment of the invention, the mesenchymal stem cells of the invention have ANKRD1, CPE, NKAIN4, LCP1, CCDC3, MAMDC2, CLSTN2, SFTA1P, EPB41L3, PDE1C compared to a comparable number of other mesenchymal stem cells. , EMILIN2, SULT1C4, TRIM58, DENND2A, CADM4, AIF1L, NTM, SHISA2, RASSF4, and ACKR3 are expressed at higher levels.

In another embodiment of the invention, the mesenchymal stem cells of the invention are selected from the group consisting of DHRS3, BMPER, IFI6, PRSS12, RDH10, and KCNE4 relative to a comparable number of other mesenchymal stem cells. express at a lower level at least one or more of the genes

Said mesenchymal stem cells and comparable numbers of other mesenchymal stem cells are derived from allogeneic tissues. More specifically, the mesenchymal stem cells are progenitor cells of mesenchymal stem cells derived from induced pluripotent stem cells.

In one embodiment of the invention, said mesenchymal stem cells are mesenchymal stem cells derived from induced pluripotent stem cells of umbilical cord tissue, and comparable numbers of other mesenchymal stem cells compared to Mesenchymal stem cells derived from umbilical cord tissue.

The present inventors named the mesenchymal stem cells differentiated from the progenitor cells of the induced pluripotent stem cell-derived mesenchymal stem cells BxC (brexogen stem cells). In the present specification, the above-mentioned "induced pluripotent stem cell-derived mesenchymal stem cells (BxC)" is also expressed as "induced pluripotent stem cell-derived mesenchymal cells".

The term “progenitor cells of induced pluripotent stem cell-derived mesenchymal stem cells” as used herein refers to cells at the stage just before the induced pluripotent stem cells are completely differentiated into mesenchymal stem cells. Mesenchymal stem cells may be regarded as a type of potent stem cell-derived mesenchymal stem cells, and refer to cells that do not express SSEA-4 protein and become complete mesenchymal stem cells upon additional culture.

The same tissue-derived (e.g., umbilical cord tissue) induced pluripotent stem cell mesenchymal stem cells (BxC) of the present invention are compared with the same tissue-derived (e.g., umbilical cord tissue) mesenchymal stem cells (MSC), There is no abnormality in the karyotype of the chromosome, and the proliferative ability is also excellent. Specifically, when the BxC of the present invention is passaged 9 times or more, it shows a difference in proliferation ability of 10 times or more compared to mesenchymal stem cells (MSCs) derived from the same tissue, and 12 times or more. No decrease in proliferation ability is observed even after repeated passages. In addition, the expression level of Ki67, which is a marker associated with cell proliferation ability, is more than twice as high in BxC as compared to MSC.

The induced pluripotent stem cell-derived mesenchymal stem cells (BxC) include Endostatin, Endothelin-1, VEGF-A, Thrombospondin-2, PlGF, PDGF-AA, beta- They secrete functional proteins such as NGF and HB-EGF in high amounts compared to comparable numbers of other mesenchymal stem cells.

Herein, said endostatin is a 20 kDa C-terminal fragment derived from naturally occurring type XVIII collagen and is reported as an anti-angiogenic agent.
Said endothelin-1, also known herein as preproendothelin-1 (PPET1), is a protein encoded by the EDN1 gene and produced in vascular endothelial cells. Endothelin-1 is known to be a potent vasoconstrictor.
As used herein, vascular endothelial growth factor A (VEGF-A (vascular endothelial growth factor A)) is a protein encoded by the VEGFA gene, and induces blood vessel growth by interacting with VEGFR1 and VEGFR2 of vascular endothelial cells. known to do.
As used herein, Thrombospondin-2 is a protein encoded by the THBS2 gene and is known to mediate cell-cell interactions or cell-matrix interactions. Although the role of thrombospondin-2 in cancer is controversial, it has been reported to modulate cell surface properties of mesenchymal stem cells and is known to be involved in cell adhesion and migration. .
As used herein, placental growth factor (PlGF) is a protein encoded by the PGF gene, and is known as a protein that plays a major role in angiogenesis during embryogenesis as a member of the VEGF subfamily. there is
As used herein, platelet-derived growth factor (PDGF-AA) is a growth factor that regulates the growth and division of cells, the formation and growth of blood vessels, and the proliferation of mesenchymal stem cells; It is known to play an important role in chemotaxis and migration.
Nerve growth factor (NGF), as used herein, is a neurotrophic factor and neuropeptide primarily involved in the growth, maintenance, proliferation, and survival of neurons. NGF is a three-protein complex in which alpha-NGF, beta-NGF, and gamma-NGF are expressed in a ratio of approximately 2:1:2. Gamma-NGF is known to act as a serine protease and cleave the N-terminus of beta-NGF to activate NGF.
As used herein, heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of proteins encoded by the HBEGF gene. HB-EGF is known to play an important role in cardiac development and vascularity, and is known to be an essential protein for epithelialization in cutaneous wound healing.

As used herein, the term "exosome" refers to a membrane vesicle having a membrane structure composed of a lipid bilayer that is secreted extracellularly by cells or present inside cells, It is present in the body fluids of most eukaryotes. The diameter of exosomes is about 30-1000 nm, and exosomes are released directly from the cell membrane when multivesicular bodies fuse with the cell membrane. Exosomes are well known to play a functional role in mediating coagulation, intercellular communication and cell-mediated immunity by transporting intracellular biomolecules such as proteins, bioactive lipids and RNA (miRNA). .

The exosome is a concept that includes microvesicles. CD63, CD81 and the like are known as exosome marker proteins. Furthermore, for example, cell surface receptors such as EGFR, signal transduction-related molecules, cell adhesion-related proteins, MSC-related antigens, heat shock proteins, Alix that is involved in vesicle formation, etc. proteins are known.

In the present invention, the exosomes isolated from the induced pluripotent stem cell-derived mesenchymal stem cells are present in the induced pluripotent stem cell-derived mesenchymal stem cells (BxC) described above, or exosomes secreted from BxC. means.
Exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells in the present invention are differentiated from progenitor cells of induced pluripotent stem cell-derived mesenchymal stem cells that do not express SSEA-4 (stage-specific embryonic antigen 4) protein. It may be one isolated from the mesenchymal stem cells.

As used herein, the term 'comprising as an active ingredient' means that exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells achieve activity for preventing or treating non-alcoholic steatohepatitis. in an amount sufficient for

In the present invention, the pharmaceutical composition contains 1 to 10000 μg, 1 to 1000 μg, 10 to 10000 μg, 10 to 1000 μg, 100 to 1000 μg of exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells based on exosome protein. It may include, but is not limited to, 10000 μg, 100-1000 μg, 50-10000 μg, 50-1000 μg, or 50-500 μg.

The term "prevention" as used in the present invention means any act of inhibiting non-alcoholic steatohepatitis or delaying progression of non-alcoholic steatohepatitis by administration of the composition of the present invention.

The term “treatment” as used herein includes (a) inhibition of progression of nonalcoholic steatohepatitis; (b) alleviation of nonalcoholic steatohepatitis; and (c) nonalcoholic steatohepatitis. means removal.

A pharmaceutical composition according to the present invention can contain a pharmaceutically acceptable carrier in addition to an active ingredient. Any pharmaceutically acceptable carrier can be included in the pharmaceutical composition of the present disclosure, so long as it is commonly used for preparing pharmaceutical compositions. Examples of pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, Including, but not limited to, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil and the like. In addition to these ingredients, the pharmaceutical compositions of the present invention can further include lubricants, moisturizers, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like.

The pharmaceutical composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally, or topically), depending on the intended method. The dose varies depending on various factors including patient's condition and body weight, severity of disease, type of formulation, administration route, and administration time, but can be appropriately selected by those skilled in the art.
A pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. In the present invention, a "pharmaceutically effective amount" means an amount sufficient to treat disease, at a reasonable benefit/risk ratio applicable to medical treatment. The effective amount may vary depending on the patient's disease type, severity, drug activity, drug sensitivity, administration time, route of administration, excretion rate, duration of treatment, concurrent drugs, and other factors well known in the medical arts. may be determined by the elements.
The pharmaceutical compositions of the present invention can be administered as individual therapeutic agents alone or in combination with other therapeutic agents. In the latter case, administration may be sequential or simultaneous. The compositions may also be administered in single or divided multiple doses. Taking all of the above factors into account, it is important to administer the lowest dose that produces the maximum effect without side effects, which is readily determinable by one skilled in the art.
Specifically, the effective amount of the pharmaceutical composition of the present invention depends on the patient's age, sex, body weight, absorption rate of the active ingredient in the body, inactivation rate, excretion rate, type of disease, and co-administration May vary by drug.

Yet another aspect of the invention relates to exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells.
Exosomes (BxC-e) isolated from mesenchymal stem cells differentiated from progenitor cells of the induced pluripotent stem cell-derived mesenchymal stem cells of the present invention (BxC-e) are exosomes having the characteristics of conventional exosomes. As demonstrated by the Examples of the present invention, the exosomes isolated from the progenitor cells of the induced pluripotent stem cell-derived mesenchymal stem cells have an excellent inhibitory effect on lipid differentiation (Fig. 2). . In addition, BxC-e of the present invention was confirmed to suppress lipogenesis, inflammation, and endoplasmic reticulum stress in steatosis-induced hepatocytes (FIGS. 5-7).

Yet another aspect of the present invention relates to a method for treating non-alcoholic steatohepatitis, comprising administering exosomes isolated from the induced pluripotent stem cell-derived mesenchymal stem cells to an individual.
The term “individual” means a subject in need of treatment for a disease, more specifically mammals such as human or non-human primates, mice, dogs, cats, horses and cows. means.

Yet another aspect of the present invention relates to the use of exosomes isolated from the induced pluripotent stem cell-derived mesenchymal stem cells for treatment of non-alcoholic steatohepatitis.
The method and therapeutic application for non-alcoholic steatohepatitis have the same components as the exosomes isolated from the induced pluripotent stem cell-derived mesenchymal stem cells of the present invention and the pharmaceutical composition containing the exosomes described above. Therefore, descriptions of the contents common to both are omitted in order to avoid undue complication of this specification.

In still another aspect of the present invention, exosomes isolated from induced pluripotent stem cells (iPSCs)-derived mesenchymal stem cells (MSCs) pretreated with a pretreatment substance are used as active ingredients It relates to a pharmaceutical composition for the prevention or treatment of non-alcoholic steatohepatitis, including as

In the present invention, induced pluripotent stem cell-derived mesenchymal stem cells are differentiated from progenitor cells of induced pluripotent stem cell-derived mesenchymal stem cells that do not express SSEA-4 (stage-specific embryonic antigen 4) protein. you can

As used herein, the term "pretreatment" refers to a process of contacting a cell culture medium supplemented with a pretreatment substance with progenitor cells of induced pluripotent stem cell-derived mesenchymal stem cells in the process of culturing progenitor cells. means.

The pretreatment may be performed by culturing the induced pluripotent stem cell-derived mesenchymal stem cells in a cell culture medium containing a pretreatment substance.
Any medium commonly used for animal cell culture can be used as the cell culture medium. For example, DMEM (Dulbecco's modification of Eagle's medium), a mixture of DMEM and F12, Eagle's MEM (Eagle's minimum essential medium), α-MEM, Iscove's MEM, 199 medium, CMRL1066, RPMI1640 , F12, F10, Way-mouth's MB752/1, McCoy's 5A, and MCDB series may be used.

Said culture may be carried out for 6-48 hours.
Specifically, the culture is carried out for 6 to 42 hours, 6 to 36 hours, 6 to 30 hours, 6 to 27 hours, 12 to 48 hours, 12 to 42 hours, 12 to 36 hours, 12 to 30 hours, 12 to 27 hours, 18-48 hours, 18-42 hours, 18-36 hours, 18-30 hours, 18-27 hours, 21-48 hours, 21-42 hours, 21-36 hours, 21-30 hours, or 21 May be performed for ˜27 hours.

The pretreatment substance is 1-(6-benzothiazolylsulfonyl)-5-chloro-1 hydrogen-indole-2-butanoic acid [1-(6-benzothiazolylsulfonyl)-5-chloro-1H-indole-2-butanoic acid], or exendin-4.
The 1-(6-benzothiazolylsulfonyl)-5-chloro-1 hydrogen-indole-2-butanoic acid [1-(6-benzothiazolylsulfonyl)-5-chloro- named "Lanifibranor" 1H-indole-2-butanoic acid] are agonists of peroxisome proliferator-activated receptors (PPARs).

Said ranifibranol may be included in the cell culture medium at a concentration of 1-100 μM.
Specifically, said ranifibranol is 1-90 μM, 1-80 μM, 1-70 μM, 1-60 μM, 1-50 μM, 1-40 μM, 1-30 μM, 10-90 μM, 10-80 μM in cell culture medium. , 10-70 μM, 10-60 μM, 10-50 μM, 10-40 μM, and 10-30 μM.

In one embodiment of the invention, ranifibranol is present in the medium at 1-1000 μM, 1-500 μM, 1-100 μM, 1-90 μM, 1-80 μM, 1-70 μM, 1-60 μM, 1-50 μM, 1- It may be added at concentrations of 40 μM, 1-30 μM, 1-20 μM, or 10 μM, but is not limited thereto.

In one embodiment of the invention, the exosomes contain 1-(6-benzothiazolylsulfonyl)-5-chloro-1 hydrogen-indole-2butanoic acid at 1000 μM, 1-500 μM, 1-100 μM, 1-90 μM , 1-80 μM, 1-70 μM, 1-60 μM, 1-50 μM, 1-40 μM, 1-30 μM, 1-20 μM, 10-90 μM, 10-80 μM, 10-70 μM, 10-60 μM, 10-50 μM, 10 Exosomes may be isolated from induced pluripotent stem cell-derived mesenchymal stem cell progenitor cells cultured in medium supplemented at concentrations of ˜40 μM, 10-30 μM, or 10 μM.

Exendin-4 of the present invention is a peptide agonist of the glucagon-like peptide (GLP) receptor. Exendin-4 stimulates insulin release and has been used clinically for treatment of type 2 diabetes and Parkinson's disease.

Said exendin-4 may be included in the cell culture medium at a concentration of 1-100 nM.
Specifically, said exendin-4 is 1-90 nM, 1-80 nM, 1-70 nM, 1-60 nM, 1-50 nM, 1-40 nM, 1-30 nM, 10-90 nM, 10-90 nM, 1-80 nM, 1-70 nM, 1-30 nM, 10-90 nM, 10 It may be included at a concentration of 80 nM, 10-70 nM, 10-60 nM, 10-50 nM, 10-40 nM, 10-30 nM, or 20 nM.

In one embodiment of the invention, the exosomes contain 1-90 nM, 1-80 nM, 1-70 nM, 1-60 nM, 1-50 nM, 1-40 nM, 1-30 nM, 10-90 nM, 10-80 nM of exendin-4. , 10-70 nM, 10-60 nM, 10-50 nM, 10-40 nM, 10-30 nM or 20 nM of induced pluripotent stem cell-derived mesenchymal stem cell progenitor cells cultured in medium supplemented with However, it is not limited to this.

In the present invention, the pharmaceutical composition comprises induced pluripotent stem cell-derived mesenchymal stem cells pretreated with 1-(6-benzothiazolylsulfonyl)-5-chloro-1 hydrogen-indole-2-butanoic acid. Exosomes isolated from progenitor cells of 1-10000 μg, 1-1000 μg, 10-10000 μg, 10-1000 μg, 100-10000 μg, 100-1000 μg, 50-10000 μg, 50-1000 μg, or 50-500 μg based on exosomal protein It may include, but is not limited to.

In the present invention, the pharmaceutical composition contains 1 to 10,000 μg, 1 to 1,000 μg of exosomes, based on exosomal protein, isolated from progenitor cells of induced pluripotent stem cell-derived mesenchymal stem cells pretreated with exendin-4. , 10-10000 μg, 10-1000 μg, 100-10000 μg, 100-1000 μg, 50-10000 μg, 50-1000 μg, or 50-500 μg.

Yet another aspect of the invention relates to exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells that have been pretreated with a pretreatment agent.
Exosomes (BxC-V37e and BxC-G63e) isolated from induced pluripotent stem cell-derived mesenchymal stem cells pretreated with the pretreatment substance of the present invention are exosomes having the properties of conventional exosomes. As demonstrated by the present examples, BxC-V37e and BxC-G63e of the present invention suppress adipogenesis, inflammation, and endoplasmic reticulum stress in steatosis-induced hepatocytes (FIGS. 5-5). Figure 10).

Yet another aspect of the present invention is a food composition for alleviating, suppressing or improving non-alcoholic steatohepatitis comprising exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells.

Still another aspect of the present invention is a food composition for alleviating, suppressing or improving non-alcoholic steatohepatitis, comprising exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells pretreated with a pretreatment substance. It is a thing.
The food composition according to the present invention has the same components as the exosomes isolated from the induced pluripotent stem cell-derived mesenchymal stem cells of the present invention described above and the pharmaceutical composition containing the same. Common contents are omitted to avoid undue complexity of the specification.

The food composition according to the present invention can include, but is not limited to, ingredients that are commonly added during food production, such as proteins, carbohydrates, lipids, nutrients, seasonings, and flavorings. .
Carbohydrates that can be contained in the food composition of the present invention include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as oligosaccharides, dextrins and cyclodextrins, xylitol, sorbitol, and erythritol. and the like, but are not limited thereto.
Flavoring agents that can be contained in the food composition according to the present invention include, but are not limited to, natural flavors such as thaumatin and stevia extract, and synthetic flavors such as saccharin and aspartame.

Still another aspect of the present invention is a method for treating non-alcoholic steatohepatitis, comprising administering exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells pretreated with the pretreatment substance to an individual. Regarding.

The term “individual” means a subject in need of treatment for a disease, more specifically mammals such as human or non-human primates, mice, dogs, cats, horses and cows. means.

Yet another aspect of the present invention relates to the therapeutic use of exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells pretreated with the pretreatment substance for non-alcoholic steatohepatitis.
The method for treating non-alcoholic steatohepatitis and its therapeutic use include exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells pretreated with the pretreatment substance of the present invention described above, and pharmaceutical exosomes containing the exosomes. Since the composition and components are common, the description of what is common to both is omitted to avoid overcomplicating the specification.

The present invention provides a preventive or therapeutic agent for non-alcoholic steatohepatitis, which contains as an active ingredient exosomes isolated from induced pluripotent stem cell-derived mesenchymal stem cells pretreated or not pretreated with a pretreatment substance. relating to scientific compositions. The exosomes of the present invention exhibit improved preventive or therapeutic effects on non-alcoholic steatohepatitis compared to exosomes isolated from conventional mesenchymal stem cells, and are useful for related research and development and commercialization. can be used.

FIG. 1A shows the average size and distribution of exosomes (BxC-e) isolated from induced pluripotent stem cell-derived mesenchymal stem cells (BxC). FIG. 1B is an electron micrograph of exosomes (BxC-e) isolated from induced pluripotent stem cell-derived mesenchymal stem cells (BxC). FIG. 2A is a photograph showing the inhibitory effect of exosomes (BxC-e) isolated from induced pluripotent stem cell-derived mesenchymal stem cells (BxC) on lipogenesis. FIG. 2B is a diagram showing the inhibitory effect of exosomes (BxC-e) isolated from induced pluripotent stem cell-derived mesenchymal stem cells (BxC) on adipogenesis. FIG. 3A shows the mean size and distribution of exosomes (BxC-V37e) isolated from ranifibranol pretreated induced pluripotent stem cell-derived mesenchymal stem cells (BxC). FIG. 3B is an electron micrograph of exosomes (BxC-V37e) isolated from ranifibranol-pretreated induced pluripotent stem cell-derived mesenchymal stem cells (BxC). FIG. 4A shows the average size and distribution of exosomes (BxC-G63e) isolated from exendin-4 pretreated induced pluripotent stem cell-derived mesenchymal stem cells (BxC). FIG. 4B is an electron micrograph of exosomes (BxC-G63e) isolated from exendin-4 pretreated induced pluripotent stem cell-derived mesenchymal stem cells (BxC). Figure 5. Exosomes (BxC-e) isolated from induced pluripotent stem cell-derived mesenchymal stem cells (BxC) and ranifibranol pretreated induced pluripotent stem cell-derived cells in steatosis-induced hepatocytes. FIG. 3 shows the lipogenesis inhibitory effect of exosomes (BxC-V37e) isolated from leaf stem cells (BxC). FIG. 6A. Exosomes (BxC-e) isolated from induced pluripotent stem cell-derived mesenchymal stem cells (BxC) in steatosis-induced hepatocytes (BxC-e) and induced pluripotent stem cell-derived cells pretreated with ranifibranol. FIG. 3 is a diagram showing the inflammation suppressive effect of exosomes (BxC-V37e) isolated from leaf stem cells (BxC). FIG. 6B. Exosomes (BxC-e) isolated from induced pluripotent stem cell-derived mesenchymal stem cells (BxC) in steatosis-induced hepatocytes (BxC-e) and induced pluripotent stem cell-derived cells pretreated with ranifibranol. FIG. 3 is a diagram showing the inflammation suppressive effect of exosomes (BxC-V37e) isolated from leaf stem cells (BxC). FIG. 7. Exosomes (BxC-e) isolated from induced pluripotent stem cell-derived mesenchymal stem cells (BxC) and ranifibranol pretreated induced pluripotent stem cell-derived cells in steatosis-induced hepatocytes. FIG. 3 is a diagram showing the endoplasmic reticulum stress (ER Stress) suppression effect of exosomes (BxC-V37e) isolated from leaf stem cells (BxC). FIG. 8A shows lipogenesis by exosomes (BxC-G63e) isolated from exendin-4 and exendin-4 pretreated induced pluripotent stem cell-derived mesenchymal stem cells (BxC) in steatosis-induced hepatocytes ( 1 is a diagram showing an inhibitory effect on lipogenesis. FIG. FIG. 8B shows lipogenesis by exosomes (BxC-G63e) isolated from exendin-4 and exendin-4 pretreated induced pluripotent stem cell-derived mesenchymal stem cells (BxC) in steatosis-induced hepatocytes ( 1 is a diagram showing an inhibitory effect on lipogenesis. FIG. FIG. 8C shows lipogenesis by exosomes (BxC-G63e) isolated from exendin-4 and exendin-4 pretreated induced pluripotent stem cell-derived mesenchymal stem cells (BxC) in steatosis-induced hepatocytes ( 1 is a diagram showing an inhibitory effect on lipogenesis. FIG. FIG. 9A. Inflammation by exosomes (BxC-G63e) isolated from exendin-4 and exendin-4 pretreated induced pluripotent stem cell-derived mesenchymal stem cells (BxC) in steatosis-induced hepatocytes. ) is a diagram showing the suppression effect. FIG. 9B shows Inflammation by exosomes (BxC-G63e) isolated from exendin-4 and exendin-4 pretreated induced pluripotent stem cell-derived mesenchymal stem cells (BxC) in steatosis-induced hepatocytes. ) is a diagram showing the suppression effect. FIG. 10. Endoplasmic reticulum stress by exendin-4 and exendin-4 pretreated induced pluripotent stem cell-derived mesenchymal stem cell (BxC) exosomes (BxC-G63e) in steatosis-induced hepatocytes. It is a figure which shows the (ER Stress) inhibitory effect. FIG. 11A shows hepatocytes of steatosis-induced mice in a normal group (Normal) treated with no substance, a control group (MCD-PBS) treated with PBS, and a BxC-V37e treated group treated with BxC-V37e ( MCD-V37e) is a photograph showing the results of hematoxylin and eosin (H&E) staining. FIG. 11B shows hepatocytes of steatosis-induced mice in a normal group (Normal) with no substance treatment, a control group (MCD-PBS) with PBS, and a BxC-V37e treatment group with BxC-V37e ( Fig. 3 is a photograph showing the results of oil red O staining of MCD-V37e). FIG. 12A shows the hepatocytes of steatosis-induced mice in a normal group (Normal) treated with no substance, a control group (MCD-PBS) treated with PBS, and a BxC-G63e treated group treated with BxC-G63e ( MCD-G63e) is a photograph showing the results of hematoxylin and eosin staining (Hematoxylin &Eosin; H&E). FIG. 12B shows hepatocytes of steatosis-induced mice in a normal group (Normal) with no substance treatment, a control group (MCD-PBS) with PBS, and a BxC-G63e-treated group with BxC-G63e ( MCD-G63e) is a photograph showing the results of oil red O staining. FIG. 13A shows hepatocytes of steatosis-induced mice in a normal group (Normal) with no substance treatment, a control group (MCD-PBS) with PBS, and BxC-V37e with BxC-V37e (MCD- V37e) Graph showing NAS scores for treatment groups. FIG. 13B shows hepatocytes of steatosis-induced mice in a normal group treated with no substance (Normal), a control group treated with PBS (MCD-PBS), and BxC-G63e treated with BxC-G63e (MCD- V63e) Graph showing NAS scores for treatment groups. FIG. 14A shows hepatocytes of steatosis-induced mice in a normal group (Normal) with no substance treatment, a control group (MCD-PBS) with PBS, and a BxC-V37e treatment group with BxC-V37e ( 1 is a photograph showing the results of immunohistochemical staining of MCD-V37e). FIG. 14B shows hepatocytes of steatosis-induced mice treated with a normal group (Normal), a control group (MCD-PBS) treated with PBS, and a BxC-G63e treated group (MCD -G63e) is a photograph showing the results of immunohistochemical staining. FIG. 15A shows hepatocytes of fibrosis-induced mice in a normal group (Normal) treated with no substance, a control group (TAA-PBS) treated with PBS, and BxC-V37e treated with BxC-V37e (TAA- V37e) Photograph showing the liver tissue of the treated group. FIG. 15B shows hepatocytes of fibrosis-induced mice in a normal group (Normal) treated with no substance, a control group (TAA-PBS) treated with PBS, and BxC-G63e treated with BxC-G63e (TAA- G63e) Photograph showing the liver tissue of the treated group. FIG. 16A shows hepatocytes of fibrosis-induced mice in a normal group (Normal) treated with no substance, a control group (TAA-PBS) treated with PBS, and BxC-V37e treated with BxC-V37e (TAA- V37e) A photograph showing the results of hematoxylin and eosin staining of the treated group. FIG. 16B shows hepatocytes of fibrosis-induced mice in a normal group (Normal) treated with no substance, a control group (TAA-PBS) treated with PBS, and BxC-G63e treated with BxC-G63e (TAA- G63e) Photograph showing the results of hematoxylin and eosin staining of the treated group. FIG. 17A shows hepatocytes of fibrosis-induced mice in a normal group (Normal) treated with no substance, a control group (TAA-PBS) treated with PBS, and BxC-V37e treated with BxC-V37e (TAA- V37e) Photograph showing picrosirius red staining results of the treated group. FIG. 17B shows hepatocytes of fibrosis-induced mice in a normal group (Normal) treated with no substance, a control group (TAA-PBS) treated with PBS, and BxC-G63e treated with BxC-G63e (TAA- G63e) Photograph showing the results of picrosirius red staining of the treated group.

Prevention of non-alcoholic steatohepatitis containing exosomes isolated from induced pluripotent stem cells (iPSCs)-derived mesenchymal stem cells (MSCs) as active ingredients , palliative, suppressive or therapeutic pharmaceutical compositions.

The present invention will be described in more detail below using examples. It will be apparent to those of ordinary skill in the art that these examples are merely illustrative of the invention and that the scope of the invention is not to be construed as limited by these examples. deaf.
Throughout this specification, "%" is used to denote the concentration of a particular substance, unless otherwise stated, solid/solid is (weight/weight) %, solid/liquid is (weight/volume) %, and liquid / liquid is (vol/vol)%.

Production Example: Isolation and Culture of Induced Pluripotent Stem Cell (iPSC)-Derived Mesenchymal Stem Cells (BxC) Cultured for 7 days. Next, from the cultured induced pluripotent stem cells, FACS is used to separate SSEA-4 (-) cells that do not express SSEA-4 (stage-specific embryonic antigen 4) protein on the cell surface, and induced pluripotent Progenitor cells of sex stem cell-derived mesenchymal stem cells were obtained. In addition, the isolated SSEA-4(-) cells were subcultured and further cultured in the same medium as above for 7 days to prepare the induced pluripotent stem cell-derived mesenchymal stem cells of the present invention. The present inventors named the induced pluripotent stem cell-derived mesenchymal stem cell BxC (brexogen stem cell).
Induced pluripotent stem cell-derived mesenchymal stem cells designated BxC were cultured in culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100X) (Gibco, Cat no. 11140-050)].

Example 1: Isolation of induced pluripotent stem cell-derived mesenchymal stem cell (BxC)-derived exosomes (BxC-e) Induced pluripotent stem cell-derived mesenchymal stem cell (hereinafter referred to as BxC) culture medium cultured in the above production example was harvested and centrifuged at 300 xg for 10 minutes to remove remaining cells and cell debris. The supernatant was collected, filtered through a 0.22 μm filter, and centrifuged at 10,000×g at 4° C. for 70 minutes using a high speed centrifuge. The centrifuged supernatant was taken again and centrifuged at 100,000×g for 90 minutes at 4° C. using an ultracentrifuge to remove the supernatant. The exosomes remaining in the lower layer were diluted with PBS (phosphate buffered saline) and used in the following experiments.

Experimental Example 1: Characterization of induced pluripotent stem cell-derived mesenchymal stem cell-derived exosomes (BxC-e) The size distribution of exosomes was confirmed using a tracking assay (NanoSight NS300, Malvern), and the morphology of exosomes was confirmed using an electron microscope.
As can be seen from FIGS. 1A and 1B, the BxC-derived exosomes of the present invention have properties as exosomes.

Experimental Example 2: Confirmation of inhibitory effect on adipogenesis of induced pluripotent stem cell-derived mesenchymal stem cell-derived exosomes (BxC-e) It was confirmed that the exosomes isolated in Example 1 had an inhibitory effect on adipogenesis. .
Human adipocytes (primary human adipocytes, ATCC, USA) were dispensed into 6-well plates and added with 1% penicillin-streptomycin and 10% CS using DMEM medium (Gibco, USA), 37 ° C., 5% It was cultured in a _CO2 incubator until it grew to a confluent state (up to 6 days).
After adipocytes were cultured for 6 days, DMEM medium (Gibco, USA) supplemented with 1% penicillin-streptomycin and 10% FBS was added with adipogenesis medium [34 μM pantothenate, Sigma, 66 μM biotin, Sigma), 0.5 mM insulin (Sigma), 1 mM dexamethasone (Sigma), and 0.05 M IBMX (Sigma)] for 5 days. DMEM medium (Gibco, USA) was then supplemented with adipogenesis medium [34 μM pantothenate, Sigma, 66 μM biotin, 0.5 mM insulin (Sigma) and 1 mM dexamethasone, Sigma]. The medium was cultured for an additional 9 days.
At this time, the negative control group (Negative Control) is adipocytes cultured for 14 days in DMEM medium supplemented with 10% FBS, vehicle control group (Vehicle control) is not treated with BxC-e, adipogenesis The BxC-e treatment group is adipocytes cultured for 14 days under adipogenic conditions containing BxC-e.
After completely removing the culture medium, the cells were washed twice with PBS, and 400 μl/well of 10% formalin solution was added to fix the cells for 1 hour. After washing with PBS, 400 μl/well of Oil Red O working solution was added to stain fat in differentiated adipocytes for 2 hours. Then, the Oil Red O working solution was removed, and the Oil Red O working solution adhering to the walls of the wells was completely removed using secondary distilled water. Alcohol (isopropyl alcohol) was added to the wells at 500 μl/well.
Absorbance was measured at 490 nm using a microplate reader (Model 680 microplate reader, Bio-Rad, USA) to quantitatively compare the amounts of lipids.
As can be seen from FIGS. 2A and 2B, the BxC-derived exosomes (BxC-e) of the present invention were found to be highly effective in suppressing lipogenesis of adipocytes.

Example 2: Separation of induced pluripotent stem cell-derived mesenchymal stem cell-derived exosomes by treatment with pretreatment substance 2-1. Induced pluripotent stem cell-derived mesenchymal stem cell-derived exosomes (BxC-V37e) treated with ranifibranol
Culture medium containing 10 μM ranifibranol [high glucose DMEM (Gibco, Cat no. 11995-065); 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acid solution (100X) (Gibco, Cat no. 11140-050)], the induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (BxC) produced in the above production example were cultured for 24 hours.
After the culture was completed, the ranifibranol-pretreated BxC was washed and cultured in culture medium supplemented with 10% exosome-depleted fetal bovine serum (FBS) for an additional 72 hours. The reason for using FBS from which exosomes have been removed is that the FBS that is normally used contains a large amount of exosomes derived from bovine serum, so that FBS-derived exosomes are mixed with exosomes secreted by cells. This is to prevent
After culturing for 72 hours, the pretreated BxC culture medium was collected and centrifuged at 300×g for 10 minutes to remove remaining cells and cell debris. The supernatant was taken, filtered using a 0.22 μm filter, and then centrifuged at 10,000×g at 4° C. for 70 minutes using a high speed centrifuge. The centrifuged supernatant was taken again, centrifuged at 100,000×g at 4° C. for 90 minutes using an ultracentrifuge, and the supernatant was removed. The exosomes remaining in the lower layer were diluted with PBS (phosphate buffered saline) and used in the following experiments.

2-2. Exendin-4-treated induced pluripotent stem cell-derived mesenchymal stem cell-derived exosomes (BxC-G63e)
Exosomes were isolated in the same manner as in Example 2-1, except that exendin-4 (20 nM) was used instead of ranifibranol in Example 2-1.

Experimental Example 3 Characterization of induced pluripotent stem cell-derived mesenchymal stem cell-derived exosomes by treatment with pretreatment substances A particle tracking assay (NanoSight NS300, Malvern) was used to confirm the size distribution of the exosomes, and electron microscopy was used to confirm the morphology of the exosomes.
As can be seen from FIGS. 3A to 4B, both BxC-derived exosomes treated with IVA337 of the present invention (FIGS. 3A and 3B) and BxC-derived exosomes treated with exendin-4 (FIGS. 4A and 4B) are exosomes. It turns out that it has a characteristic.

Experimental Example 4: Evaluation of therapeutic activity of induced pluripotent stem cell-derived mesenchymal stem cell-derived exosomes (BxC-V37e) treated with ranifibranol for non-alcoholic steatohepatitis exosomes (BxC-e) isolated in Example 1 And the exosomes (BxC-V37e) isolated in Example 2-1 were tested as follows.

4-1. Steatosis induction
Materials and reagents
DMEM (Dulbecco's modified Eagle's medium) medium was purchased from Hyclone (Pittsburgh, PA, USA), and FBS (fetal bovine serum) was purchased from Gibco (Grand Island, NY, USA). Free fatty acid-free BSA (Fatty acid-free bovine serum albumin), palmitate, and oleate were purchased from Sigma (St. Louis, MO, USA).
Culture of human hepatocyte line (HepG2)
A human liver cell line (HepG2, ATTC) was cultured in DMEM medium containing 10% FBS (Gibco) and 1% penicillin-streptomycin at 37° C. and 5% CO ₂ . After the cultured cells were dispensed into a 6-well plate at a certain number (5×10 ⁵ cells/1000 μl/well), the cells were completely attached to the wells in a perfect morphology and reached 95% confluence. Incubate until
Production of free fatty acid (FFA)
(1) Preparation of 1 M palmitate and 1 M oleate stock solutions Solutions were prepared at 60° C. using 70% ethanol and deionized distilled water (ddH ₂ O) as solvents and passed through a 0.2 μm filter. It was sterilized after filtering using.
(2) Preparation of 1% (w/v) BSA solution containing no free fatty acid The solution was prepared using deionized distilled water as a solvent, then sterilized and refrigerated at 4°C.
(3) Preparation of mixed fatty acid in which palmitate and oleate are mixed so that the concentration is 100 mM The 1% (w / v) BSA solution containing no free fatty acid produced in (2) above is used as a solvent. The concentration of the fatty acid produced in (1) above was adjusted to 100 mM. [10 µl of 1 M palmitate prepared in (1) + 290 µl (33 mM) of 1% (w/v) BSA] and [10 µl of 1 M oleate prepared in (1) + 140 µl of 1% (w/v) BSA (66 mM)] were mixed in a 1:1 volume ratio in a 70° C. heatblock.
(4) Preparation of mixed free fatty acids (FFA) at a final concentration of 1 mM 10 μl of the mixed fatty acid 100 mM solution prepared in (3) above (2:1 oleate and palmitate containing 1% (w/v) BSA) ) was mixed with 990 μl of serum-free DMEM medium containing 1% penicillin-streptomycin to prepare a final FFA concentration of 1 mM.
(5) Preparation of BSA control solution The 1% (w/v) BSA prepared in (2) above was used as a control solution.
Induction of steatosis
The medium of the HepG2 cells that had reached 95% confluency was discarded, and the cells were washed once with PBS. Then, 1 ml of the 1 mM mixed free fatty acid produced in (4) above was added to each well of 6 wells. Vehicle Control received serum-free medium treated with the same amount of vehicle. All treatments were performed overnight (16 hours).

4-2. Evaluation of therapeutic activity for nonalcoholic steatohepatitis The HepG2 cells treated for 16 hours were washed once with PBS, and 100 μg of BxC-e isolated in Example 2 and 100 μg of BxC-V37e isolated in Example 2 were added. After treatment with 1 ml of serum-free medium for 24 hours, the following experiments were carried out.

[1] Suppressive effect on lipogenesis Human hepatocytes were degraded by adding 1 ml of Trizol solution per well of a 6-well plate. After adding 200 μl of chloroform and vortexing, the mixture was centrifuged at 4° C. and 12,000 rpm for 15 minutes. After transferring the supernatant to a new tube, it was mixed with 500 μl of isopropanol. After inverting 50 times and leaving on ice for 5 minutes, it was centrifuged at 12,000 rpm and 4° C. for 10 minutes. The supernatant was removed, and 1 ml of 70% ethanol was added to the remaining pellet, followed by centrifugation at 12,000 rpm and 4°C for 5 minutes. After removing the ethanol, the tube containing the RNA pellet was dried at room temperature to make it invisible. The RNA pellet was dissolved using nuclease free water. Nanodrop was used to measure the concentration of extracted RNA samples at 260 nm and 280 nm wavelengths, and RT premix was used to synthesize cDNA.
The synthesized cDNA was subjected to real-time PCR (real-time-polymerase chain reaction) using synthesized primers (Cosmogene Tech) (see Table 1 below) to monitor FABP4 mRNA expression.

As can be seen from FIG. 5, the expression level of the FABP4 gene related to lipogenesis decreased in the group of steatosis-induced human hepatocytes treated with BxC-V37e compared to the untreated group. did. From this, it was confirmed that BxC-V37e has a high inhibitory activity against adipogenesis.

[2] Suppressive effect on inflammation Using the same method as in Example 4-[1], changes in TNF-α and MCP1 mRNA expression were confirmed. The primers used are shown in Table 2 below.

As can be seen from FIGS. 6A and 6B, in the group in which steatosis-induced human hepatocytes were treated with BxC-V37e, the expression levels of TNF-α and MCP-1 expressed during inflammation induction were significantly decreased compared to the group that did not. This confirmed the excellent anti-inflammatory activity of BxC-V37e in inflammatory conditions induced by adipogenesis.

[3] Effect of Suppressing Endoplasmic Reticulum Stress (ER Stress) Changes in CHOP mRNA expression were confirmed using the same method as in Example 4-[1]. The primers used are shown in Table 3 below.

As can be seen from FIG. 7, in the group in which steatosis-induced human hepatocytes were treated with BxC-V37e, the CHOP gene expression level related to endoplasmic reticulum stress was significantly higher than in the untreated group. decreased to From this, it was confirmed that BxC-V37e has a very excellent inhibitory activity against endoplasmic reticulum stress induced by lipogenesis.

Experimental Example 5: Evaluation of therapeutic activity of induced pluripotent stem cell-derived mesenchymal stem cell-derived exosomes (BxC-G63e) treated with exendin-4 and exendin-4 isolated in Example 2-2 Experiments were performed on exosomes (BxC-G63e) as follows.

5-1. Steatosis induction
Materials and reagents
DMEM (Dulbecco's modified Eagle's medium) medium was purchased from Hyclone (Pittsburgh, PA, USA), and FBS (fetal bovine serum) was purchased from Gibco (Grand Island, NY, USA). Fatty acid-free bovine serum albumin (BSA) free of free fatty acids, palmitate (palmitate) and oleate (oleate) were purchased from Sigma (St. Louis, MO, USA). .
Human liver cell line (HepG2) culture
A human liver cell line (HepG2, ATTC) was cultured in DMEM medium containing 10% FBS (Gibco) and 1% penicillin-streptomycin at 37° C. and 5% CO ₂ . After the cultured cells were dispensed into a 6-well plate at a certain number (5×10 ⁵ cells/1000 μl/well), the cells were completely adhered to the wells with intact morphology, and the cells reached 95% confluence. incubated until
Production of Free Fatty Acid (FFA)
(1) Preparation of 1 M palmitate and 1 M oleate stock solutions Solutions were prepared at 60° C. using 70% ethanol and deionized distilled water (ddH ₂ O) as solvents and passed through a 0.2 μm filter. It was sterilized after filtering using.
(2) Preparation of 1% (w/v) BSA solution containing no free fatty acids The solution was prepared using deionized distilled water as a solvent, then sterilized and refrigerated at 4°C.
(3) Preparation of mixed fatty acid in which palmitate and oleate are mixed so that the concentration is 100 mM The 1% (w / v) BSA solution containing no free fatty acid produced in (2) above is used as a solvent. The concentration of the fatty acid produced in (1) above was adjusted to 100 mM. [10 µl of 1 M palmitate prepared in (1) + 290 µl (33 mM) of 1% (w/v) BSA] and [10 µl of 1 M oleate prepared in (1) + 140 µl of 1% (w/v) BSA (66 mM)] were mixed in a 1:1 volume ratio in a 70° C. heatblock.
(4) Preparation of mixed free fatty acids (FFA) at a final concentration of 1 mM 10 μl of the mixed fatty acid 100 mM solution prepared in (3) above (2:1 oleate and palmitate containing 1% (w/v) BSA) ) was mixed with 990 μl of serum-free DMEM medium containing 1% penicillin-streptomycin to prepare a final FFA concentration of 1 mM.
(5) Preparation of BSA control solution The 1% (w/v) BSA prepared in (2) above was used as a control solution.
Steatosis induction
The medium of the HepG2 cells that had reached 95% confluence was discarded, and the cells were washed once with PBS, and then 1 ml of the 1 mM mixed free fatty acid produced in (4) above was added to each well of 6 wells. Vehicle Control received serum-free medium treated with the same amount of vehicle. All treatments were performed overnight (16 hours).

5-2. Non-alcoholic steatohepatitis therapeutic activity evaluation The HepG2 cells treated for 16 hours were washed once with PBS, and 100 μg of 20 nM exendin-4 or 100 μg of BxC-G63e isolated in Example 2 was added to 1 ml of serum-free medium for 24 hours. After time treatment, the following experiments were performed.

[1] Suppressive effect on lipogenesis Human hepatocytes were degraded by adding 1 ml of Trizol solution per well of a 6-well plate. After adding 200 μl of chloroform and vortexing, the mixture was centrifuged at 4° C. and 12,000 rpm for 15 minutes. After transferring the supernatant to a new tube, it was mixed with 500 μl of isopropanol. After inverting 50 times and leaving on ice for 5 minutes, it was centrifuged at 12,000 rpm and 4° C. for 10 minutes. The supernatant was removed, and 1 ml of 70% ethanol was added to the remaining pellet, followed by centrifugation at 12,000 rpm and 4°C for 5 minutes. After removing the ethanol, the tube containing the RNA pellet was dried at room temperature to make it invisible. Nuclease-free water was used to dissolve the RNA pellet. Nanodrops were used to measure the concentration of extracted RNA samples at 260 nm and 280 nm wavelengths, and RT premix was used to synthesize cDNA.
The synthesized cDNA was subjected to real time-polymerase chain reaction (PCR) using synthesized primers (Cosmogene Tech) (see Table 4 below) to monitor changes in mRNA expression of FABP4, ACC1 and SREBP1.

As can be seen from FIGS. 8A-8C, in the group in which steatosis-induced human hepatocytes were treated with BxC-G63e, FABP4, ACC1, and SREBP1 gene expression levels related to lipogenesis were significantly lower than those in the treatment. decreased compared to the group without. From this, it was confirmed that BxC-G63e has a very excellent inhibitory function on adipogenesis.

[2] Suppressive effect on adipogenesis (Inflammation) Using the same method as in Example 5-[1], changes in TNF-α and IL-10 mRNA expression were confirmed. The primers used are shown in Table 5 below.

As can be seen from FIGS. 9A and 9B, the expression levels of TNF-α and IL-10 were confirmed in the group in which steatosis-induced human hepatocytes were treated with BxC-G63e. The expression level of TNF-α, which has a pro-inflammation function, was significantly decreased, and the expression level of IL-10, which has an anti-inflammation function, was significantly increased compared to the control group. This confirmed that the anti-inflammatory effect of BxC-G63e is very good in the inflammatory situation induced by adipogenesis.

[3] Effect of Suppressing Endoplasmic Reticulum Stress (ER Stress) Changes in CHOP mRNA expression were confirmed using the same method as in Example 5-[1]. The gene primers used are shown in Table 3 above.
As can be seen from FIG. 10, in the group in which steatosis-induced human hepatocytes were treated with BxC-G63e, the CHOP gene expression level related to endoplasmic reticulum stress was significantly higher than in the untreated group. decreased to From this, it was confirmed that BxC-G63e has a very excellent inhibitory activity against endoplasmic reticulum stress induced by adipogenesis.

Experimental Example 6: Evaluation of therapeutic activity of BxC-V37e and BxC-G63e for non-alcoholic steatohepatitis using MCD-diet mouse model Exosomes (BxC-V37e) isolated in Example 2-1 and Example 2 -2 isolated exosomes (BxC-G63e) were tested as follows.

6-1. Steatosis induction in MCD diet mice
Rearing environment for MCD diet mice
C57BL/6NHsd male mice were placed at a temperature of 23±3° C., a relative humidity of 55±15%, a ventilation rate of 10-20 times/hr, a lighting time of 12 hours (lights on at 8:00 am and lights off at 8:00 pm), and an illuminance of 150-300 Lux. Environmental conditions were measured periodically while rearing in the set environment.
MCD diet salary
Six-week-old C57BL/6NHsd male mice were fed MCD diets for 5 days followed by normal diets for 2 days, alternating MCD diets and normal diets ad libitum for 12 weeks.
Preparation and administration of dosing test substance
Each test article was prepared by diluting BxC-G63e or BxC-V37e in PBS. A protein amount of 100 μg/mouse to 400 μg/mouse of the test substance was intravenously administered once a day and 3 times a week for 4 weeks. A 6-week-old C57BL/6NHsd male mouse was restrained in a holder, and the test substance was slowly injected through the tail vein at a rate of 1 mL/min using a syringe fitted with a 26-gauge needle.

6-2. Evaluation of therapeutic activity for non-alcoholic steatohepatitis The livers of C57BL/6NHsd male mice to which the test substance was administered were excised, photographed, and weighed. The right lobe was fixed with a 10% neutral buffered formalin solution, and the left lobe was flash-frozen with liquid nitrogen and used for the following experiments.

[1] Suppressive effect on lipogenesis
Histopathological examination
After common tissue processing steps such as trimming, dehydration, paraffin embedding, and sectioning, fixed liver tissue was used to prepare specimens for pathological examination. The samples were stained with hematoxylin and eosin (H&E) and oil red O (oil red O), and histopathological changes were observed using an optical microscope (Olympus BX53, Japan).
NAS scoring
For Microvesicular steatosis, Macrovesicular steatosis, Hepatocyte hypertrophy as a percentage of the total area affected in the visual field at 40x to 100x magnification under the microscope. was graded from 0 to 3 points based on Inflammation was compared between groups after scoring from 0 to 3 at 100x magnification for 5 lesions in a 100x field.
As can be seen from FIGS. 11A and 11B, and FIGS. 12A and 12B, steatosis was induced by the MCD diet, and the groups treated with BxC-G63e or BxC-V37e decreased the size of the lipid droplets. and numbers were observed to be significantly reduced compared to the control group not treated with test substance.
In addition, as can be seen from FIG. 13A or FIG. 13B, NAS scores for microvesicular steatosis, macrovesicular steatosis, and hypertrophy of hepatocytes all decreased. From this, it was confirmed that BxC-G63e or BxC-V37e has a very excellent inhibitory effect on adipogenesis.

[2] Suppressive effect on inflammation After general tissue processing processes such as trimming, dehydration, paraffin embedding, and sectioning, specimens for pathological examination were prepared using fixed liver tissue. . The sample was reacted with anti-TNF-α as a primary antibody, and then further reacted with a secondary antibody specific thereto, and then observed for changes in TNF-α protein expression.
As can be observed from Figures 14A and 14B, the MCD diet was used to induce inflammation, and the BxC-G63e or BxC-V37e treated groups showed significantly higher levels of TNF-α than the PBS treated control group. It was confirmed that the protein expression level was significantly reduced. In addition, as can be seen from FIG. 13A, the inflammation score was decreased in the group treated with BxC-V37e. Therefore, it was confirmed that BxC-G63e or BxC-V37e has a very high inhibitory activity against inflammation.

Experimental Example 7: Evaluation of non-alcoholic steatohepatitis therapeutic activity of BxC-V37e and BxC-G63e using TAA mouse model Exosomes (BxC-V37e) isolated in Example 2-1 and Example 2-2 The exosomes isolated in (BxC-G63e) were tested as follows.

7-1. Induction of fibrosis in TAA mice
Rearing environment for TAA diet mice
C57BL/6 male mice were exposed to a temperature of 23±3° C., a relative humidity of 55±15%, a ventilation rate of 10-20 times/hr, a lighting time of 12 hours (lights on at 8:00 am and lights off at 8:00 pm), and an illuminance of 150-300 Lux. Environmental conditions were measured periodically while rearing in the set environment.
TAA drug administration
Six-week-old C57BL/6 male mice were administered TAA at a concentration of 200 mg/kg once daily, three times weekly for 12 weeks.
Preparation and administration of dosing test substance
Each test article was prepared by diluting BxC-G63e or BxC-V37e in PBS. The test substance was subcutaneously administered at 400 μg/animal once a day, three times a week for 4 weeks, and at the same time, TAA at a concentration of 200 mg/kg was administered once a day, twice a week for 4 weeks. For injection of substances, the administration site was disinfected with 70% alcohol. After creating a space between the skin and the muscle by pulling the skin of the right thigh of the mouse with the thumb and index finger, an insulin syringe was inserted into the subcutaneous space between the thumb and index finger from the front of the animal and insulin was administered.

7-2. Evaluation of therapeutic activity for non-alcoholic steatohepatitis The livers of C57BL/6 male mice to which the test substance was administered were excised, photographed, and weighed. The right lobe was fixed with a 10% neutral buffered formalin solution, and the left lobe was flash-frozen with liquid nitrogen and used for the following experiments.

[1] Suppressive effect on fibrosis
Histopathological examination
After common tissue processing steps such as trimming, dehydration, paraffin embedding, and sectioning, fixed liver tissue was used to prepare specimens for pathological examination. The specimens were stained with hematoxylin & eosin (H&E) and picrosirius red, and histopathological changes were observed using an optical microscope (Olympus BX43, Japan).
As can be seen from FIGS. 15A and 15B, it was confirmed that the surface of liver tissue in the TAA-PBS group was less smooth than in the normal group without any treatment. It could be observed that treatment with BxC-G63e or BxC-V37e significantly restored liver tissue.
In addition, as can be confirmed from FIGS. 16A and 16B, as a result of H&E staining, it was observed that TAA-damaged liver tissue was significantly restored by BxC-G63e or BxC-V37e.
As can be seen from FIGS. 17A and 17B, picrosirius red staining showed that the amount of collagen accumulated in liver tissue by TAA was significantly reduced by BxC-G63e or BxC-V37e. rice field.
In short, it was confirmed that BxC-G63e or BxC-V37e has excellent inhibitory activity against fibrosis

Summary Summarizing the foregoing, BxC-e, BxC-V37e, and BxC-G63e of the present invention also suppress adipogenesis, inflammation, and endoplasmic reticulum stress in steatosis-induced hepatocytes, thus non-alcoholic It finds very advantageous use in the prevention or treatment of steatohepatitis.

The present invention provides a non-alcoholic fat containing, as an active ingredient, exosomes derived from mesenchymal stem cells differentiated from induced pluripotent stem cell-derived mesenchymal stem cell progenitor cells pretreated or not pretreated with a pretreatment substance. The present invention relates to a composition for preventing or treating sexual hepatitis.

Claims (12)

Prevention of non-alcoholic steatohepatitis containing exosomes isolated from induced pluripotent stem cells (iPSCs)-derived mesenchymal stem cells (MSCs) as active ingredients , palliative, suppressive or therapeutic pharmaceutical compositions. The induced pluripotent stem cell-derived mesenchymal stem cells according to claim 1, which are derived from progenitor cells of induced pluripotent stem cell-derived mesenchymal stem cells that do not express SSEA-4 (stage-specific embryonic antigen 4) protein. A pharmaceutical composition of 2. The pharmaceutical composition according to claim 1, wherein the induced pluripotent stem cells are human-derived induced pluripotent stem cells. Exosomes isolated from progenitor cells of induced pluripotent stem cell-derived mesenchymal stem cells. 5. The induced pluripotent stem cell-derived mesenchymal stem cells according to claim 4, which are derived from progenitor cells of induced pluripotent stem cell-derived mesenchymal stem cells that do not express SSEA-4 (stage-specific embryonic antigen 4) protein. A pharmaceutical composition of Non-alcoholic steatohepatitis containing as an active ingredient exosomes isolated from induced pluripotent stem cells (iPSCs)-derived mesenchymal stem cells (MSCs) pretreated with a pretreatment substance A pharmaceutical composition for prevention, alleviation, suppression or treatment of (Non-alcoholic steatohepatitis). 7. The induced pluripotent stem cell-derived mesenchymal stem cells according to claim 6, which are derived from progenitor cells of induced pluripotent stem cell-derived mesenchymal stem cells that do not express SSEA-4 (stage-specific embryonic antigen 4) protein. A pharmaceutical composition of The pretreatment substance is 1-(6-benzothiazolylsulfonyl)-5-chloro-1 hydrogen-indole-2-butanoic acid [1-(6-benzothiazolylsulfonyl)-5-chloro-1H-indole-2-butanoic acid] and exendin-4 (Exendin-4). 7. The pharmaceutical composition according to claim 6, wherein the induced pluripotent stem cells are human-derived induced pluripotent stem cells. Relief of non-alcoholic steatohepatitis containing exosomes isolated from induced pluripotent stem cells (iPSCs)-derived mesenchymal stem cells (MSCs) as active ingredients , control or amelioration food compositions. 11. The induced pluripotent stem cell-derived mesenchymal stem cells according to claim 10, which are derived from progenitor cells of induced pluripotent stem cell-derived mesenchymal stem cells that do not express SSEA-4 (stage-specific embryonic antigen 4) protein. food composition. The food composition according to claim 10, wherein the induced pluripotent stem cells are human-derived induced pluripotent stem cells.

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